FBXO36 Antibody

What is FBXO36 and what cellular functions does it perform?

FBXO36 is a 188 amino acid protein that contains one forty amino acid F-box region, making it a member of the F-box protein family. F-box proteins are critical components of the SCF (Skp1-CUL-1-F-box protein) type E3 ubiquitin ligase complex and are involved in substrate recognition and recruitment for ubiquitination. These proteins regulate essential cellular processes including cell cycle progression, immune response, signaling cascades, and developmental programs by targeting proteins such as cyclins, cyclin-dependent kinase inhibitors, IkB-å and β-catenin for degradation via the proteasome after ubiquitination. Functioning as a component of the SCF complex, FBXO36 specifically recognizes and binds to select phosphorylated proteins, thereby promoting their ubiquitination and subsequent degradation .

What are the key specifications of commercially available FBXO36 antibodies?

FBXO36 antibodies are available in various formats with different specifications as detailed in the table below:

Research applications require careful selection of the appropriate antibody specifications based on the experimental design, target species, and detection method .

How should I design experiments to effectively validate FBXO36 antibody specificity?

Effective antibody validation requires a multi-faceted approach:

• Positive and negative controls: Include tissues or cell lines known to express FBXO36 (positive control) and those with minimal expression (negative control).

• Multiple detection methods: Cross-validate results using different techniques (Western blot, immunofluorescence, ELISA) to ensure consistent detection.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

• Knockout/knockdown validation: Compare staining in wild-type cells versus FBXO36 knockout or knockdown samples to verify specificity.

• Cross-reactivity assessment: Test against tissues from multiple species to confirm predicted reactivity patterns.

• Dilution optimization: Perform a titration series to identify the optimal antibody concentration that maximizes signal-to-noise ratio.

These methodological steps are critical for establishing antibody specificity and reliability before proceeding with experimental applications 11.

What control groups should be included when studying FBXO36 protein interactions?

When studying FBXO36 protein interactions, rigorous experimental controls should include:

• Input control: Sample of cell lysate before immunoprecipitation to verify target protein expression.

• Isotype control: Immunoprecipitation with an irrelevant antibody of the same isotype to identify non-specific binding.

• Other F-box protein controls: Include closely related F-box proteins (e.g., FBXO4, FBXO22) as comparative controls, as research has shown differential behavior between F-box proteins in certain cellular contexts .

• No-antibody control: Processing samples without antibody to identify non-specific protein binding to beads.

• Reciprocal co-immunoprecipitation: Validate interactions by immunoprecipitating with antibodies against suspected interacting partners.

• Cell cycle synchronization controls: When studying cell cycle-dependent interactions, include synchronized cells at different cell cycle stages, as research has shown some F-box proteins exhibit cell cycle-dependent modifications while FBXO36 does not show similar band shifts during mitosis .

This comprehensive control strategy minimizes false positives and provides robust validation of protein-protein interactions .

How can I investigate FBXO36's role in the ubiquitin-proteasome pathway?

To investigate FBXO36's role in the ubiquitin-proteasome pathway, implement the following methodological approach:

• Substrate identification:

  • Perform IP-MS (immunoprecipitation followed by mass spectrometry) to identify potential FBXO36 substrates

  • Validate interactions with co-immunoprecipitation using FBXO36 antibodies

  • Confirm direct binding through in vitro binding assays with purified proteins

• Ubiquitination assays:

  • Conduct in vivo ubiquitination assays by co-expressing FBXO36, potential substrates, and HA-tagged ubiquitin

  • Perform in vitro ubiquitination using purified components of the SCF complex including FBXO36

  • Use proteasome inhibitors (MG132) to accumulate ubiquitinated proteins for detection

• Substrate stability assays:

  • Measure protein half-life in cells with normal vs. depleted FBXO36 using cycloheximide chase experiments

  • Monitor substrate levels after FBXO36 overexpression or knockdown

  • Assess substrate phosphorylation states, as FBXO36 recognizes phosphorylated proteins for ubiquitination

• Structure-function analysis:

  • Create F-box domain mutants to disrupt SCF complex formation

  • Identify substrate recognition domains through deletion mapping

  • Analyze how mutations affect substrate ubiquitination and degradation

This systematic approach provides comprehensive insights into FBXO36's specific role in targeting proteins for proteasomal degradation .

What approaches can be used to study differential expression of FBXO36 in disease models?

To investigate FBXO36 expression in disease models, implement a multi-level analysis strategy:

• Transcript-level analysis:

  • qRT-PCR to quantify FBXO36 mRNA expression across disease states and control tissues

  • RNA-seq to identify correlation patterns with other genes in disease pathways

  • Single-cell RNA-seq to identify cell type-specific expression changes

• Protein-level detection:

  • Western blotting for semi-quantitative analysis of FBXO36 protein levels

  • Optimize antibody dilutions (1:300-5000 for WB) based on expression levels

  • Immunohistochemistry/immunofluorescence to visualize spatial distribution in tissues

    • Use appropriate dilutions (IHC-P: 1:200-400; IF: 1:50-200)

    • Include proper controls for antibody specificity

• Functional correlations:

  • Correlate FBXO36 expression with levels of known or suspected target proteins

  • Perform interaction studies (co-IP) to identify disease-specific binding partners

  • Monitor cellular phenotypes associated with altered FBXO36 levels

• Model systems:

  • Compare expression between normal and disease tissues in human samples

  • Utilize mouse models to track expression changes during disease progression

  • Employ cell line models that recapitulate disease states for mechanistic studies

This comprehensive approach enables meaningful correlation of FBXO36 expression patterns with disease pathogenesis .

How can I troubleshoot weak or non-specific signal when using FBXO36 antibodies in Western blot?

When troubleshooting Western blot issues with FBXO36 antibodies, consider this methodical approach:

• Antibody-specific optimization:

  • Titrate antibody concentration (start with manufacturer's recommendation of 1:300-5000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider alternative FBXO36 antibodies targeting different epitopes (N-terminal vs. internal regions)

• Sample preparation enhancement:

  • Enrich for cytoplasmic fraction (FBXO36 is primarily cytoplasmic)

  • Use freshly prepared lysates to prevent protein degradation

  • Add protease/phosphatase inhibitors to preserve protein integrity

  • Consider using denaturation conditions that maintain FBXO36 epitope accessibility

• Detection system optimization:

  • Use enhanced chemiluminescence (ECL) substrates with appropriate sensitivity

  • Consider signal amplification systems for low-abundance detection

  • Optimize exposure times based on FBXO36's expected expression level

• Non-specific binding reduction:

  • Increase blocking time/concentration (5% BSA may be more effective than milk for some antibodies)

  • Add 0.1-0.5% Tween-20 in washing steps

  • Use more stringent washing conditions (higher salt concentration)

  • Pre-absorb antibody with proteins from non-relevant species

• Protein size verification:

  • FBXO36 is expected at approximately 22.1 kDa , although post-translational modifications may alter migration

  • Unlike other F-box proteins like Fbxo6, FBXO36 does not show band shifts during mitosis

These systematic adjustments address the most common technical challenges encountered with FBXO36 detection .

What are the critical considerations for preserving FBXO36 antibody integrity and performance?

To maintain optimal FBXO36 antibody performance over time, implement these evidence-based storage and handling practices:

• Temperature management:

  • Store antibodies at -20°C for long-term storage as recommended by manufacturers

  • Avoid repeated freeze-thaw cycles that can lead to antibody degradation

  • Prepare small working aliquots to minimize freeze-thaw events

  • Allow antibodies to equilibrate to room temperature before opening to prevent condensation

• Buffer considerations:

  • Maintain antibodies in manufacturer's buffer (typically 0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol)

  • Add carrier proteins (BSA) if diluting stock antibody

  • Avoid introducing microbial contamination by using sterile technique

• Handling protocols:

  • Centrifuge vials briefly before opening to collect liquid at the bottom

  • Use sterile pipette tips when handling antibody solutions

  • Minimize exposure to light for fluorophore-conjugated antibodies

  • Document lot numbers and performance characteristics for reproducibility

• Quality control measures:

  • Perform regular validation tests on stored antibodies

  • Include positive controls in each experiment to verify antibody activity

  • Monitor signal-to-noise ratio over time to detect potential degradation

• Shipping and transfer considerations:

  • Maintain cold chain during transport (typically shipped at 4°C)

  • Check for signs of improper handling upon receipt (freeze-thaw, warming)

  • Allow sufficient equilibration time after transport before use

Proper antibody management is critical for experimental reproducibility and reliability, particularly for antibodies targeting proteins like FBXO36 that may be expressed at lower levels .

How should researchers interpret contradictory results between different detection methods for FBXO36?

When faced with contradictory results between different detection methods for FBXO36, implement this structured analytical approach:

• Technique-specific considerations:

  • Western blot detects denatured protein, revealing size and abundance

  • Immunohistochemistry/immunofluorescence shows spatial distribution but may be affected by fixation methods

  • ELISA quantifies native protein but may miss conformational changes

• Antibody epitope analysis:

  • Compare the epitope regions targeted by different antibodies

  • Antibodies targeting different regions (N-terminal vs. AA 32-81 vs. AA 66-165) may yield different results

  • Post-translational modifications may mask certain epitopes in specific contexts

• Expression level assessment:

  • FBXO36 may be expressed at different levels across tissues and cell types

  • Sensitivity differences between methods may explain discrepancies

  • Use quantitative methods (qPCR for mRNA) to complement protein detection

• Cross-reactivity evaluation:

  • Verify if contradictory results stem from cross-reactivity with related proteins

  • F-box proteins share structural similarities that could lead to non-specific binding

  • Compare results against knockout/knockdown controls when possible

• Integrated data interpretation strategy:

  • Weight results based on methodological strengths and limitations

  • Consider biological context when interpreting conflicting data

  • Use orthogonal approaches (functional assays) to resolve contradictions

  • Document all contradictions transparently in research reports

What are the key considerations when analyzing FBXO36's potential interacting partners?

When analyzing potential FBXO36 interacting partners, apply this comprehensive analytical framework:

• Interaction strength and specificity analysis:

  • Classify interactions as strong, moderate, or weak based on detection consistency

  • Compare interaction profiles with other F-box proteins (FBXO4, FBXO22, FBXO6) to identify specific vs. common partners

  • Assess interaction dependency on F-box domain by using truncation mutants

• Functional categorization of interactors:

  • Group interacting proteins by biological function (cell cycle, signaling, etc.)

  • Identify enriched pathways using gene ontology analysis

  • Analyze protein domains that might mediate interactions with FBXO36

• Post-translational modification analysis:

  • Examine phosphorylation states of interacting proteins, as FBXO36 recognizes phosphorylated targets

  • Determine whether interactions are dependent on specific modifications

  • Monitor modification changes throughout the cell cycle

• Temporal dynamics assessment:

  • Unlike FBXO6, FBXO36 does not show modification (band shifts) during mitosis

  • Investigate whether interactions are cell cycle-dependent despite this lack of modification

  • Compare interaction profiles across different cellular states (stress, differentiation)

• Validation hierarchy:

  • Rank interactions based on validation strength (detection by multiple methods)

  • Prioritize interactions confirmed by reciprocal co-IP and functional assays

  • Consider evolutionary conservation of interactions across species

This structured approach provides a comprehensive framework for distinguishing biologically relevant FBXO36 interactions from experimental artifacts .

What are promising research avenues for understanding FBXO36's role in cellular homeostasis?

Several promising research directions can advance our understanding of FBXO36's role in cellular homeostasis:

• Substrate identification and characterization:

  • Implement proteome-wide approaches (BioID, IP-MS) to identify novel FBXO36 substrates

  • Characterize the structural and sequence determinants that mediate substrate recognition

  • Develop computational models to predict potential FBXO36 targets based on known substrates

• Regulatory mechanisms investigation:

  • Explore how FBXO36 expression and activity are regulated at transcriptional and post-translational levels

  • Identify kinases responsible for phosphorylating FBXO36 substrates to prime them for recognition

  • Investigate whether FBXO36 itself undergoes regulated degradation

• Tissue-specific functions:

  • Map FBXO36 expression across tissues using antibodies validated for immunohistochemistry (1:200-400 dilution)

  • Generate conditional knockout models to study tissue-specific phenotypes

  • Explore differential substrate targeting across tissue types

• Pathological implications:

  • Unlike FBXO6, which promotes cancer cell growth and disrupts chromosome segregation when overexpressed , FBXO36's potential role in pathological conditions remains unexplored

  • Investigate FBXO36 expression in cancer and other diseases

  • Assess correlations between FBXO36 levels and disease progression or therapeutic response

• Therapeutic potential assessment:

  • Evaluate FBXO36 as a potential drug target for conditions involving dysregulated protein degradation

  • Develop strategies to modulate specific FBXO36-substrate interactions

  • Screen for small molecules that can inhibit or enhance FBXO36 activity

These research directions could significantly advance our understanding of FBXO36's physiological and pathological roles .

How can advanced imaging techniques enhance our understanding of FBXO36 dynamics?

Advanced imaging techniques offer powerful approaches to investigate FBXO36 dynamics:

• Super-resolution microscopy applications:

  • Employ STORM or PALM imaging to visualize FBXO36 distribution at nanoscale resolution

  • Use structured illumination microscopy (SIM) to examine co-localization with SCF complex components

  • Optimize immunofluorescence protocols with validated antibody dilutions (IF: 1:50-200)

• Live-cell imaging strategies:

  • Generate fluorescent protein-tagged FBXO36 constructs for real-time visualization

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure FBXO36 mobility

  • Use FRET-based sensors to detect FBXO36-substrate interactions in living cells

• Multi-channel co-localization analysis:

  • Simultaneously visualize FBXO36, SCF complex components, and potential substrates

  • Quantify co-localization coefficients under different cellular conditions

  • Track changes in localization patterns throughout the cell cycle

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence microscopy with EM to visualize FBXO36 in the context of cellular ultrastructure

  • Implement immunogold labeling for precise subcellular localization

  • Map FBXO36 distribution relative to proteasomes and other degradation machinery

• Tissue imaging applications:

  • Apply multiplexed immunofluorescence to analyze FBXO36 expression in complex tissues

  • Use tissue clearing techniques for 3D visualization of FBXO36 distribution

  • Implement in situ proximity ligation assays to detect protein-protein interactions in intact tissues